The family Retroviridae comprises a variety of enveloped RNA viruses, such as endogenous retroviruses, leukemia viruses and immunodeficiency viruses, all of which have as their replicative strategy, the essential step of reverse transcription of the virion RNA into linear double-stranded DNA and the subsequent integration of this DNA into the genome of a cell. All retroviruses contain genes for the proteins Gag, Pol, Env, and (often) Pro, while more complex retroviruses, such as HIV-1, contain additional regulatory proteins.
Human immunodeficiency virus 1 (HIV-1) is the causative agent of acquired immunodeficiency syndrome (AIDS). HIV-1 is transmissible in samples of body fluids including blood, plasma, and semen, and as such, it is important to determine whether a sample may be contaminated with HIV-1 before administering the sample in a medical procedure or using the sample to prepare a medical product. For protection of patients who might otherwise receive a sample of HIV-1-infected body fluid, it is particularly important to detect the presence of the virus in the sample to prevent its use in such procedures or in products.
Early-developed tests for detecting HIV-1 relied on detecting anti-HIV-1 antibodies in a sample of body fluid. However, because there is a delay between the time a subject is infected with HIV-1 and the time that the subject develops antibodies against HIV-1, these early-developed tests were prone to false negative results. As such, new tests were developed that could detect HIV-1 nucleic acid in a sample from a subject shortly after the subject has been infected.
Traditional methods for detecting nucleic acids include Southern Blots for detecting DNA, and Northern Blots and RNase mapping for detecting RNA. However, these methods have limited sensitivity. With the advent of reverse transcription (“RT”), the polymerase chain reaction (“PCR”), and automated DNA sequencing, scientists and clinicians were presented with much more sensitive assays for detecting DNA and RNA. For example, methods of performing RT, PCR, reverse transcription followed by PCR (“RT-PCR”), and sequencing are described in U.S. Pat. No. 6,593,086; U.S. Pat. No. 6,569,647; U.S. Pat. No. 6,532,276; U.S. Pat. No. 6,518,019; U.S. Pat. No. 6,500,620; U.S. Pat. No. 6,495,350; U.S. Pat. No. 6,379,191; U.S. Pat. No. 6,274,320; U.S. Pat. No. 6,127,115; U.S. Pat. No. 5,962,665; U.S. Pat. No. 5,876,924; and U.S. Pat. No. 5,618,703.
“Reverse transcriptase” describes a class of polymerases characterized as RNA-dependent DNA polymerases in that they use an RNA template to synthesize a DNA molecule. Historically, reverse transcriptases have been used to reverse-transcribe mRNA into cDNA. However, reverse transcriptases can be used to reverse-transcribe other types of RNAs such as viral genomic RNA or viral sub-genomic RNA. Standard reverse transcriptases include Maloney Murine Leukemia Virus Reverse Transcriptase (MoMuLV RT) and Avian myoblastosis virus (AMV). These enzymes have 5′→3′ RNA-dependent DNA polymerase activity, 5′→3′ DNA-dependent DNA polymerase activity, and RNase H activity. However, unlike many DNA-dependent DNA polymerases, these enzymes lack 3′→5′ exonuclease activity necessary for “proofreading,” (i.e., correcting errors made during transcription). After a DNA copy of an RNA has been prepared, the DNA copy may be subjected to various DNA amplification methods such as PCR. Reverse transcription and PCR can be combined in a single assay referred to as RT-PCR.
The PCR process provides a method for amplifying small amounts of DNA, (theoretically a single molecule), into easily detectable amounts. (See U.S. Pat. Nos. 4,683,195 and 4,683,202, which describe the PCR process.) The standard method for PCR requires a template molecule (i.e., the molecule to be amplified), primers that anneal to the template molecule, a DNA dependent DNA polymerase, and a buffer that, in particular, includes nucleotides. The primers are allowed to anneal to the template molecule and the DNA-dependent DNA polymerase extends the primers to create a nascent ds-DNA copy of the template molecule. The sample is then heated to denature the nascent ds-DNA and the primers are again allowed to anneal. As such, the nascent ds-DNA can function as a template molecule for further rounds of ds-DNA synthesis. Because the primers are usually present in excess, the reaction is only limited by the concentration of the template molecule. As the amount of available template increases, the amount of synthesized DNA increases exponentially.
Because the PCR process typically involves heating the sample to denature the ds-DNA, the PCR reaction was not practical until the discovery of thermostable DNA-dependent DNA polymerases. By using a thermostable DNA-dependent DNA polymerase, fresh polymerase need not be added after the sample is heated to denature the dsDNA. The PCR process typically utilizes a DNA polymerase that is stable up to about 93°-95° C.
Reverse transcription and PCR can be used to detect a variety of pathogens including HIV-1. HIV-1 belongs to the family of retroviruses and the subfamily of lentiviruses. Like all retroviruses, HIV-1 is an enveloped, single-stranded (ss), positive-sense (+) RNA virus, and the genome of HIV-1 consists of a dimer of two of the single-stranded RNA molecules. During infection, HIV-1 converts its RNA genome into a DNA copy (i.e., a “provirus”) by using its native reverse transcriptase. HIV-1 inserts this DNA copy of its genome into the infected cell's DNA by using a viral enzyme called “integrase.” As such, the genome of HIV-1 may be present as an RNA copy or DNA copy within the infected cell.
RT-PCR can be used to detect HIV-1 in blood or tissue samples and to diagnose HIV-1 infection at any time after infection. Diagnosis by RT-PCR is more sensitive and can detect infection earlier than the standard HIV antibody tests (usually an ELISA).
Human T-cell Leukemia Virus types I and II (i.e., HTLV-I and HTLV-II, respectively, also called human T-cell lymphotropic virus types I and II), are members of the family of retroviruses and the subfamily of lentiviruses. HTLV-I has transforming activity in vitro and is etiologically linked to adult T-cell leukemia, which is known to be endemic in several parts of the world. HTLV-II is another retrovirus having transforming capacity in vitro, and has been isolated from a patient with a T-cell variant of hairy cell leukemia. The diagnosis of HTLV-I infection is usually based on serum antibody response to HTLV-I peptide antigens. This usually involves an initial screening assay to identify HTLV-I antibodies, based on an enzyme immunoassay (EIA) with HTLV-I virion peptides. Of those individuals tested for HTLV-I and HTLV-II using standard assays for blood screening, about 0.5-0.05% test positive. However, about 4 out of 5 of the positive results are false positives. Therefore, positive sera is typically tested in a confirmatory assay, such as Western blotting or radioimmunoprecipitation, which detect antibody reactivity with specific HTLV-I peptide antigens.
RT-PCR provides a useful approach for detecting HTLV I and II in peripheral blood mononuclear cells from infected individuals. Early detection of HTLV-I and HTLV-II infection is useful because it is associated with lung pathologies (e.g., increased incidence of pneumonia and acute bronchitis).
As such, improved methods for diagnosing retroviral infections in humans such as those caused by HIV and HTLV is important in treating infected individuals and in controlling the spread of disease.